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1. Journal of Natural Sciences Research www.iiste.org
ISSN 2224-3186 (Paper) ISSN 2225-0921 (Online)
Vol.3, No.9, 2013
38
Species Distribution and Virulence Factors of Coagulase Negative
Staphylococci Isolated From Clinical Samples From the
University of Benin Teaching Hospital, Edo State, Nigeria.
Azuka Azih and Idahosa Enabulele
Department of Microbiology, Faculty of Life Sciences, University of Benin, Benin City, Nigeria.
*E-mail of the corresponding author: zukkycare@yahoo.com
Abstract
Coagulase negative staphylococci (CoNS) have become a major public health problem due to an increase in
invasive procedures and antibiotic resistance. The prevalence and some virulence factors of CoNS species
isolated from clinical samples obtained from a hospital in Benin-City, Nigeria was investigated. A total of 79
clinical samples were collected from clinical specimens over a six month period in a non consecutive manner
from a teaching hospital in Nigeria. Samples were cultured on MacConkey agar, Cystine –Lysine-Electrolyte-
Deficient agar (CLED) and blood agar (Oxoid Ltd Basingstoke UK). Identification of the various CoNS species
was done using Microbact staph ID system (Oxoid product). Determination of hemolysin production was done in
blood agar plates while slime detection was carried out using the modified congo red agar method. The most
commonly isolated species were S. haemolyticus (28.3%), S. epidermidis (26.7%) and S. saprophyticus
(18.33%). Others were S. simulans (10%), S. xylosus (10%), S. chromogenes (15%) and S. schleiferi (1.67%).
Hemolysis on blood agar plates was observed in 34.2% of CoNS with S. haemolyticus (58.8%) predominating
followed by S. epidermidis (25%). Slime production was positive in 75.95% of all CoNS species isolated. All
isolates of S. simulans (100%) and S. xylosus (100%) were positive for slime production followed by S.
epidermidis (93.7%) and S. haemolyticus (70.6%). Due to the increasing medical significance of CoNS, these
organisms should not be ignored or classified as mere contaminants.
Keywords: Coagulase negative Staphylococcus, virulence factors, infections, slime, hemolysin.
INTRODUCTION
Coagulase negative staphylococci (CoNS) have been identified as the etiological agent in various
infections and are among the microorganisms most frequently isolated in nosocomial infections (Cunha et al.,
2006). This has generated a new research interest in them. CoNS have been implicated in neonatal septiceamia
(Ghelbi et al., 2008), bacterial eye infections (Ogbolu et al., 2011) infective endocarditis (Patel et al., 2000), and
prosthetic joint infections (Trampuz and Zimmerli, 2005). Although Staphylococcus epidermidis is the CoNS
most commonly isolated in the clinical microbiology laboratory, other CONs species are also increasingly being
associated with infections. Other species known to cause infections includes Staphylococcus xylosus,
Staphylococcus hominis, Staphylococcus schleifieri, Staphylococcus saprophyticus (urinary tract infections in
immunocompetent women), Staphylococcus lugdunensis (implicated in sepsis), and S. haemolyticus which has
been associated with endocarditis and osteomyelitis (Venkatesh et al., 2006). Evidence indicates that
pathogencity might be related to the production of an extracellular polysaccharide known as slime that permits
these microorganisms to adhere to smooth plastic surfaces, colonizing catheters, prosthetic heart valves,
pacemakers and Joint prosthesis (Vogel et al., 2000) and tissue surfaces.
Many CoNS also produce several lipases, proteases and other exoenzymes which possibly contribute to
the persistence of CoNS in the host and may degrade host tissues (Otto, 2004). Hemolysins were also produced
by isolates of S .epidermidis, S. haemolyticus, S. warneri. Hemolysin binds to susceptible cells leading to rapid
release of vital molecules. Most importantly, they lyse red blood cells for release of free iron taken up by the
bacteria. S. haemolyticus has also been reported to produce hemolysin and is often multiple antibiotic resistant
(Gunn and Davis, 1988). Although S. epidermidis is believed to account for most of the infections caused by
CoNS, the specific infecting CoNS species remains undetermined in most clinical laboratories.
CoNS species identification which is necessary in order to establish epidemiological trends, confirm
treatment failures and determine the cause of specific infections in the community. Understanding the
mechanisms of pathogenesis will help in the development of preventive and control measures. This research is
aimed at determining the prevalence of CoNS, identifying the various species and some of the virulence factors
of CoNS isolated from clinical samples in our community.
MATERIALS AND METHOD
Sample collection
Seventy-nine samples from different clinical conditions were collected from a teaching hospital over a period of

2. Journal of Natural Sciences Research www.iiste.org
ISSN 2224-3186 (Paper) ISSN 2225-0921 (Online)
Vol.3, No.9, 2013
39
6 months (April to September, 2011). The clinical samples were from infected wound (14), urine (13) exudates
from male genital infections (10), swab samples from female genital infections (10), tonsilitis and pharyngitis
(8), urinary catheter (2), blood (suspected septiceamia) (4), eye infections (6), otitis media (1), infected burns (6)
and other clinical samples (5).
Culture and identification of Coagulase Negative Staphylococci Species
All samples except urine samples were cultured on Blood agar plates, MacConkey agar, Robertson Cooked Meat
agar (RCM) and incubated aerobically at 37o
C for 24 hours. Urine samples were cultured on Cystine Lysine
Electrolyte Deficient (CLED) medium and MacConkey agar plates at 37o
C for 24 hours. Suspected
staphylococcal colonies were identified by Gram staining, catalase and coagulase test. CoNS were detected by
using the slide and tube coagulase tests. Identification of CoNS species was done using the MICROBACT
Staphylococcus identification system 12S (Oxoid Ltd Basingstoke UK). The system uses a combination of sugar
utilization and colorimetric enzyme detection substrates. Each kit contains Staph 12S test strips each consisting
of 12 wells containing different dehydrated substrates, suspending media consisting of buffering agents and
peptones for preparing the inoculum, a holding tray, an organism report form and a color interpretation chart.
The color chart was used as a guide for interpreting reactions and the results entered into a computer aided
software to produce probable species.
Hemolysin Production
Production of hemolysin was determined on blood agar plates incubated at 37o
C for 24 hours. A
positive result was indicated by the formation of hemolytic zones around the colonies.
Slime formation
Assessment of slime formation was determined using the Congo red agar method (Freeman et al. 1989).
Several colonies of a single isolate were inoculated on plates of the medium and incubated aerobically for
24hours at 37o
C. A positive result was indicated by black colonies. Non-slime producers usually remained pink.
RESULTS
A total of 105 CoNS isolates from different clinical samples were obtained following either tube or
slide coagulase tests. With both tube and slide coagulase, 79 isolates were coagulase negative and the remaining
26 coagulase positive which was discarded.
CoNS were most commonly isolated from infected wounds (17.7%), followed by urine from cases of
urinary tract infections (16.5%) and least isolated from ear infections (1.26%) (Table I). Infected wounds were
mainly from surgical wounds, diabetic foot ulcer and prosthetic devices.
S. haemolyticus (28.3%) was the most commonly isolated CoNS closely followed by S. epidermidis
(26.7%), then S. saprophyticus (18.3%). The remaining species were distributed between S. simulans (10%), S.
xylosus (10%), S. chromogenes (5%) and S. schleiferi (1.67%). (Table II)
S. haemolyticus also had the highest number of heamolytic strains (58.8%) followed by S. epidermidis
(25%), S. saprophyticus (25%), S. xylosus (50%), S. simulans (50%) and non speciated CoNS (10.53%). (Table
III)
Slime formation by CoNS species is shown in Table IV. Most of the isolates were positive for slime
production (75.95%). Predominantly, S. xylosus, S. schleiferi, S. simulans (100%), S. epidermidis (93.7%) and S.
heamolyticus (70.6%) also had a good number of slime producing strains.
DISCUSSION
CoNS, once considered as members of the normal flora of the human body are now known to be a major cause
of opportunistic infections.
The highest number of CoNS isolates were from found wounds samples (17.7%) followed by urine
(16.5%). This is in line with the reports of Vuong and Otto. (2002) who reported CoNS as the major aetiologic
agent in wound infections and urinary tract infections.
S. haemolyticus (28.33%), S. epidermidis (26.67%) and S. saprophyticus (18.33%) were the most
commonly isolated CoNS species and this result is not significantly different from other reports where S.
epidermidis was the highest isolated followed by S. haemolyticus in clinical infections (Begum et al., 2011).
Kloos and Bannermann (1995) also reported that S. epidermidis and S. saprophyticus as the most isolated CoNS
species in clinical infections. Other isolates were S. simulans (10%), S. xylosus (10%), S. chromogenes (5%) and
S. schleiferi (1.67%). This is similar to a work done by Adeleye et al. (2010) and Akinkunmi and Lamikanra,
(2010) that isolated similar CoNS species from clinical samples. S. epidermidis was the most isolated specie of
CoNS implicated in wound infections and this is in accordance with similar works earlier reported (Duran et al.,
2010). Lark et al. (2001) reported that the skin of 80-90% of people is colonized with S. epidermidis and that
most CoNS infections are acquired from patients own flora. In Nigeria, CoNS is one of the common causes of
infections of open fractures in wounds and delay in wound debridement is a major predisposing factor to wound

3. Journal of Natural Sciences Research www.iiste.org
ISSN 2224-3186 (Paper) ISSN 2225-0921 (Online)
Vol.3, No.9, 2013
40
infection (Ikem et al., 2004). S. simulans has been associated with cases of osteomyelitis and prosthetic joint
infections. It is common in the normal flora and primarily acquired through contact with domestic animals
(Kline, 2010). CoNS were also reported in burns in this study and this can be attributed to the
immunocompromising effect of burn wounds (Shahnaz and Mohammadali, 2011).
Prevalence of S. saprophyticus, S. epidermidis and S. haemolyticus has also been reported in urine
samples (Kumari et al., 2001) Pathogenicity of S. saprophyticus in urinary tract could be attributed to the fact
that it has the ability to adhere to uroepithelial cells (Raz et al., 2004). S. haemolyticus grows in moderate
numbers on human skin and is one of the few CoNS species found in urethra or periurethra of men and women.
(Gunn and Davis, 1988) and this could be a source of infection. They’ve been implicated in UTI in males of all
ages leading to epididymitis, urethritis and prostratitis. Isolation of S. epidermidis from catheterized patients was
not surprising as they have been reported to be the most prominent cause of intravascular associated catheter
infection (Rupp and Archer, 1994). S. epidermidis also produce slime which enables them adhere to these
surfaces leading to biofilm formation. CoNS have also been isolated in catheter associated urinary tract
infections in Nigeria (Taiwo and Aderounmu, 2006).
Infections of the female genitals investigated in this study were of pelvic inflammatory disease and
cervicitis. S. haemolyticus and S. saprophyticus were isolated from these sites. The isolation of S. epidermidis
and S. simulans from samples of scrotal aspirate, urethral swab and semen is similar to reports of Barcs et al
(1989). The implication of S. saprophyticus, S. epidermidis and S. simulans in ear, nose and throat (ENT)
infections is in agreement with the reports of Yildirim et al., 2004 who isolated CoNS from 45.8% of chronic
sinusitis and Akinjogunla and Enabulele (2010) who implicated CoNS in 42.9% of cases of otitis media. These
bacteria may proliferate to cause infections under conditions that defeat the normal mucocillary defense
mechanism. S. simulans however has not been reported in the past in throat infections. More reports are
necessary to ascertain its status as an opportunistic pathogen.
S. xylosus was the only CoNS species isolated from blood and it has earlier been reported by Begum et
al. (2011) as an etiologic agent of bacteraemia. S. xylosus has been reported to produce a delta-hemolysin that
gave synergistic, complete hemolysis of washed human, sheep, and ox blood cells in an area of beta-lysin
activity from strains of S. aureus and S. intermedius. S. chromogenes which is rarely a human pathogen was also
isolated in this study and it has earlier been implicated in blood stream infections of some patients in Nigeria
(Adeyemi et al., 2010). Implication of S. epidermidis in eye infections have also been reported earlier in patients
from south western Nigeria where they were incriminated in endopthalmitis, chronic blepharitis and corneal
ulcers (Ogbolu et al., 2011).
Researchers have earlier reported hemolysin production by CoNS (Cunha et al., 2006, Akinjogunla and
Enabulele, 2010). It was mostly observed in S. haemolyticus (37.6%) and lowest in S. simulans (11.1%) and S.
xylosus (11.1%). Predominance of hemolysin production by S. haemolyticus is not surprising as it has been
reported to be its major virulence factor (Gunn and Davis, 1988). Hemolysin production has also been earlier
reported in S. saprophyticus by Raz et al. (2005) as well as S. epidermidis.
Biofilm formation is thought to play an important role in the pathogenesis of CoNS induced infections. A high
percentage (76%) of CoNS species isolated in this report was positive for slime production. Slime production
was observed in 93.7% of S. epidermidis isolates and this is similar to a work reported by Ishak et al. (1985)
where slime producing strains of S. epidermidis represented 13 of 14 (92.86%) isolates. The virulence factors of
S. saprophyticus include adherence to uroepithelial cells by means of a surface associated protein and this could
be attributed to slime production observed in S. saprophyticus (54.5%). Other virulence factors of S.
saprophyticus are reported to include lipoteichoic acid, hemagglutinin and hemolysin (Raz et al., 2005). Some
researchers have also observed adherence in S. schleiferi which is presumed to be evidence of slime production
(Herbert, 1990).
CONCLUSION
Coagulase negative staphylococci regarded as normal commensals of the human body have also become one of
the most important pathogens in clinical infections especially among immunocompromised patients with medical
devices (Vuong and Otto, 2002). Because of its increasing medical significance which has also been confirmed
in this survey, routine laboratory identification of this group of pathogens is necessary in order to establish
epidemiological trends and in infection control.
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4. Journal of Natural Sciences Research www.iiste.org
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Vol.3, No.9, 2013
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TABLE 1: Occurence of CoNS isolated from various clinical samples
Source No of Isolates (%)
Wounds infected 14(17.72%)
Infected burns 6(7.59%)
Exudates from male genitals infections 10(12.66%)
Swab samples from female genitals
infections
10(12.66%)
Tonsilitis and pharyngitis 8(10.13%)
Urine from urinary tract infections 13(16.5%)
Urinary catheter 2(2.53%)
Blood (suspected septiceamia) 4(5.06%)
Eye infections 6(7.59%)
Otitis media 1(1.26%)
Other clinical samples 5(6.33%)
Total 79(100%)

6. Journal of Natural Sciences Research www.iiste.org
ISSN 2224-3186 (Paper) ISSN 2225-0921 (Online)
Vol.3, No.9, 2013
43
TABLE 2: Distribution of CoNS species among the various clinical samples
Source of samples
(n)
Staphylococc
us.epidermidi
s
S.saprophyti
cus
S.heamolyti
cus
S.
xylosus
S.
simulans
S.chromoge
nes
S.
schleife
ri
Non-
speciated
CoNS
A Wounds
infected(14)
4 1 4 1 1 - - 3
B Burns infected (6) 3 1 1 - - - - 1
C Urine infected (13) 2 4 3 - - 1 - 3
D Urinary catheter (2) 1 - - - - - - 1
E Swab samples from
female genital
infections (10)
- 3 6 - - - - 1
f Exudates from
male genital
infections (10)
3 - 1 1 2 - - 3
g
.
Throat (tonsillitis
and pharyngitis) (8)
1 2 - - 3 - - 2
H Eye infections (6) 1 - - - - 2 1 2
I Suspected
septicemia (4)
- - - 4 - - - -
J Otitis media (1) 1 - - - - - - -
k Other clinical
samples (5)
- - 1 - - 1 - 3
Total number 16(26.70%) 11(18.30%) 17(28.3%) 6(10.00
%)
6(10.00
%)
3(5%) 1(1.67
%)
19(24.05
%)
of each Isolate (%)
n= number of samples
TABLE 3: Prevalence of hemolysin production in Coagulase negative Staphylococci
Isolates Total no of isolates No of hemolytic strains (%)
Staphylococcus epidermidis 16 4 (25%)
S. saprophyticus 11 4 (36.36%)
S. haemolyticus 17 10 (58.82%)
S. simulans 6 3 (50%)
S. xylosus 6 3 (50%)
S. chromogenes 3 -
S. schleiferi 1 -
Non- speciated CoNS 19 2 (10.53%)
TABLE 4: Slime formation by Coagulase negative Staphylococci
CoNS species No. of isolates No of slime producing isolates
(%)
Staphylococcus haemolyticus 17 12 (70.58 %)
S. epidermidis 16 15(93.75%)
S. saprophyticus 11 6(54.55%)
S. simulans 6 6 (100%)
S. xylosus 6 6 (100%)
S. chromogenes 3 1 (33.33%)
S. schleiferi
Non-speciated CoNS
1
19
1 (100%)
12(63.15%)
Total 79 60(75.95%)

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